15-Deoxy-Δ¹²,¹⁴ prostaglandin J₂ reduces the formation of atherosclerotic lesions in apolipoprotein E knockout mice

Aim

15-Deoxy-Δ^12,14 Prostaglandin J₂ (15d-PGJ₂) is a ligand of peroxisome proliferator-activated receptor γ (PPARγ) having diverse effects such as the differentiation of adipocytes and atherosclerotic lesion formation. 15d-PGJ₂ can also regulate the expression of inflammatory mediators on immune cells independent of PPARγ. We investigated the antiatherogenic effect of 15d-PGJ₂.

Methods

We fed apolipoprotein (apo) E-deficient female mice a Western-type diet from 8 to 16 wk of age and administered 1 mg/kg/day 15d-PGJ₂ intraperitoneally. We measured atherosclerotic lesions at the aortic root, and examined the expression of macrophage and inflammatory atherosclerotic molecules by immunohistochemical and real-time PCR in the lesion.

Results

Atherosclerotic lesion formation was reduced in apo E-null mice treated with 15d-PGJ₂, as compared to in the controls. Immunohistochemical and real-time PCR analyses showed that the expression of MCP-1, TNF-α, and MMP-9 in atherosclerotic lesions was significantly decreased in 15d-PGJ₂ treated mice. The 15d-PGJ₂ also reduced the expression of macrophages and RelA mRNA in atherosclerotic lesions.

Conclusion

This is the first report 15d-PGJ₂, a natural PPARγ agonist, can improve atherosclerotic lesions in vivo. 15d-PGJ₂ may be a beneficial therapeutic agent for atherosclerosis.     

7beta-hydroxy-epiandrosterone modulation of 15-deoxy-delta12,14-prostaglandin J2, prostaglandin D2 and prostaglandin E2 production from human mononuclear cells

7β-hydroxy-epiandrosterone (7β-OH-EPIA) has been shown to be cytoprotective in various organs including the brain. It has also been shown that prostaglandin D₂ (PGD₂) and its spontaneous metabolite 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) are also cytoprotective. It is possible that these prostaglandins derived from circulating mononuclear cells may mediate the actions of 7β-OH-EPIA. The aim of this study, therefore, was to ascertain the effect of 7β-OH-EPIA (in the absence or presence of tumour necrosis factor-α (TNF-α)), a pro-inflammatory stimulus, on the biosynthesis of PGD₂, PGE₂ and 15d-PGJ₂ from human mononuclear cells. Prostaglandins were measured by enzyme immunoassay (EIA). 7β-OH-EPIA alone induced a concentration-dependant increase in the production of PGD₂. TNF-α increased PGD₂ levels which were enhanced by 7β-OH-EPIA. 7β-OH-EPIA increased 15d-PGJ₂ levels both in the absence and presence of TNF-α. 7β-OH-EPIA alone had no effect on PGE₂ biosynthesis but suppressed TNF-α-induced PGE₂ circa 50%. 7β-OH-EPIA also increased the level of free arachidonic acid and radiolabelled prostaglandins in cells pre-incubated with radiolabelled arachidonic acid, indicating that the increase may occur via the enhanced release of substrate arachidonic acid. 7β-OH-EPIA did not affect levels of the anti-inflammatory cytokine IL-10 indicating that this is an unlikely mechanism by which 7β-OH-EPIA induces its actions but more likely exerts its effects via the production of cytoprotective prostaglandins.     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

Background

In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.

Methods

Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP₁-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF_2α-and PGE₂-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.

Results

Epidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF_2α-and PGE₂-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF_2α-and PGE₂-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP₁-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.

Conclusion

The results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.     

The Anti-inflammatory Prostaglandin 15-Deoxy-Δ12,14-PGJ2 Inhibits CRM1-dependent Nuclear Protein Export

The signaling molecule 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) has been described as the “anti-inflammatory prostaglandin.” Here we show that substrates of the nuclear export receptor CRM1 accumulate in the nucleus in the presence of 15d-PGJ₂, identifying this prostaglandin as a regulator of CRM1-dependent nuclear protein export that can be produced endogenously. Like leptomycin B (LMB), an established fungal CRM1-inhibitor, 15d-PGJ₂ reacts with a conserved cysteine residue in the CRM1 sequence. This covalent modification prevents the formation of nuclear export complexes. Cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of LMB and 15d-PGJ₂, demonstrating that the same single amino acid is targeted by the two compounds. Inhibition of the CRM1 pathway by endogenously produced prostaglandin and/or exogenously applied 15d-PGJ₂ may contribute to its anti-inflammatory, anti-proliferative, and anti-viral effects.     

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) Induces Cell Death Through Caspase-independent Mechanism in A172 Human Glioma Cells

15-Deoxy-^Δ12,14-prostaglandin J₂ (15d-PGJ₂) is a naturally occurring cyclopentenone metabolite of prostaglandin D₂ (PGD₂) and is known as a specific potent ligand for the peroxisome proliferators activator receptor-γ (PPARγ). 15d-PGJ₂ inhibits cell growth and induces apoptosis in a number of different cancer cells. However, the underlying mechanism by which 15d-PGJ₂ induces cell death remains to be defined. The present study was undertaken to determine the effect of 15d-PGJ₂ on cell death in A172 human glioma cells. 15d-PGJ₂ caused reactive oxygen species (ROS) generation. 15d-PGJ₂-induced ROS production and cell death were prevented by the antioxidant N-acetylcysteine. Activation of mitogen-activated protein kinases (MAPK) was not observed in cells treated with 15d-PGJ₂ and inhibitors of MAPK subfamilies also were not effective in preventing 15d-PGJ₂-induced cell death. 15d-PGJ₂ treatment caused mitochondrial dysfunction, as evidenced by depolarization of mitochondrial membrane potential. 15d-PGJ₂ induced caspase activation at 24 h of treatment, but the 15d-PGJ₂-induced cell death was not prevented by caspase inhibitors. The antiapoptotic protein XIAP levels and release of apoptosis inducing factor (AIF) into the cytosol were not altered by 15d-PGJ₂ treatment. Taken together, these findings indicate that 15d-PGJ₂ triggers cell death through a caspase-independent mechanism and ROS production and disruption of mitochondrial membrane potential play an important role in the 15d-PGJ₂-induced cell death in A172 human glioma cells.     

Prostaglandin D2-glycerol ester decreases carrageenan-induced inflammation and hyperalgesia in mice

Pain is one of the cardinal signs of inflammation and is present in many inflammatory conditions. Therefore, anti-inflammatory drugs such as NSAIDs also have analgesic properties. We previously showed that prostaglandin D₂-glycerol ester (PGD₂-G), endogenously produced by cyclooxygenase-2 from the endocannabinoid 2-arachidonoylglycerol, has anti-inflammatory effects in vitro and in vivo that are partly mediated by DP1 receptor activation. In this work, we investigated its effect in a model of carrageenan-induced inflammatory pain. PGD₂-G decreased hyperalgesia and edema, leading to a faster recovery. Moreover, PGD₂-G decreased carrageenan-induced inflammatory markers in the paw as well as inflammatory cell recruitment. The effects of PGD₂-G were independent from metabolite formation (PGD₂ and 15d-PGJ₂-G) or DP1 receptor activation in this model. Indeed PGD₂ delayed recovery from hyperalgesia while 15d-PGJ₂-G worsened the edema. However, while PGD₂-G decreased hyperalgesia in this model of inflammatory pain, it had no effect when tested in the capsaicin-induced pain model. While the targets mediating the effects of this bioactive lipid in inflammatory pain remain to be elucidated, our findings further support the interest of anti-inflammatory lipid mediators in the management of inflammatory pain.     

Dexamethasone Protects Neonatal Hypoxic-Ischemic Brain Injury via L-PGDS-Dependent PGD2-DP1-pERK Signaling Pathway

Background and Purpose

Glucocorticoids pretreatment confers protection against neonatal hypoxic-ischemic (HI) brain injury. However, the molecular mechanism remains poorly elucidated. We tested the hypothesis that glucocorticoids protect against HI brain injury in neonatal rat by stimulation of lipocalin-type prostaglandin D synthase (L-PGDS)-induced prostaglandin D₂ (PGD₂)-DP₁-pERK mediated signaling pathway.

Methods

Dexamethasone and inhibitors were administered via intracerebroventricular (i.c.v) injections into 10-day-old rat brains. Levels of L-PGD₂, D prostanoid (DP₁) receptor, pERK1/2 and PGD₂ were determined by Western immunoblotting and ELISA, respectively. Brain injury was evaluated 48 hours after conduction of HI in 10-day-old rat pups.

Results

Dexamethasone pretreatment significantly upregulated L-PGDS expression and the biosynthesis of PGD₂. Dexamethasone also selectively increased isoform pERK-44 level in the neonatal rat brains. Inhibitors of L-PGDS (SeCl₄), DP₁ (MK-0524) and MAPK (PD98059) abrogated dexamethasone-induced increases in pERK-44 level, respectively. Of importance, these inhibitors also blocked dexamethasone-mediated neuroprotective effects against HI brain injury in neonatal rat brains.

Conclusion

Interaction of glucocorticoids-GR signaling and L-PGDS-PGD₂-DP₁-pERK mediated pathway underlies the neuroprotective effects of dexamethasone pretreatment in neonatal HI brain injury.     

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Osteolytic Breast Cancer Bone Metastasis and Estrogen Deficiency-Induced Bone Loss

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ₂ on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ₂ dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ₂ suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ₂ blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ₂ exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ₂ substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ₂ prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ₂ may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.     

Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation

Background

Bradykinin (BK) induces angiogenesis by promoting vessel permeability, growth and remodeling. This study aimed to demonstrate that the B2R antagonist, fasitibant, inhibits the BK pro-angiogenic effects.

Methodology

We assesed the ability of fasibitant to antagonize the BK stimulation of cultured human cells (HUVEC) and circulating pro-angiogenic cells (PACs), in producing cell permeability (paracellular flux), migration and pseocapillary formation. The latter parameter was studied in vitro (matrigel assay) and in vivo in mice (matrigel plug) and in rat model of experimental osteoarthritis (OA). We also evaluated NF-κB activation in cultured cells by measuring its nuclear translocation and its downstream effectors such as the proangiogenic ciclooxygenase-2 (COX-2), prostaglandin E-2 and vascular endothelial growth factor (VEGF).

Principal findings

HUVEC, exposed to BK (1–10 µM), showed increased permeability, disassembly of adherens and tight-junction, increased cell migration, and pseudocapillaries formation. We observed a significant increase of vessel density in the matrigel assay in mice and in rats OA model. Importantly, B2R stimulation elicited, both in HUVEC and PACs, NF-κB activation, leading to COX-2 overexpression, enhanced prostaglandin E-2 production. and VEGF output. The BK/NF-κB axis, and the ensuing amplification of inflammatory/angiogenic responses were fully prevented by fasitibant as well as by IKK VII, an NF-κB. Inhibitor.

Conclusion

This work illustrates the role of the endothelium in the inflammation provoked by the BK/NF-κB axis. It also demonstates that B2R blockade by the antaogonist fasibitant, abolishes both the initial stimulus and its amplification, strongly attenuating the propagation of inflammation.     

15-Deoxy-Δ-prostaglandin J2 impairs the functions of histone acetyltransferases through their insolubilization in cells

The cyclopentenonic prostaglandin 15-deoxy-Δ^12,14-PG J₂ (15d-PGJ₂) is a metabolite derived from PGD₂. Although 15d-PGJ₂ has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor γ (PPARγ), the functions are not fully understood. In order to examine the effect of 15d-PGJ₂ on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ₂. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-κB-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ₂ attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ₂ on the function of HATs was independent of PPARγ, because PPARγ agonists could not mimick 15d-PGJ₂ and PPARγ antagonists did not inhibit 15d-PGJ₂.     

Candida albicans induces cyclo-oxygenase 2 expression and prostaglandin E2 production in synovial fibroblasts through an extracellular-regulated kinase 1/2 dependent pathway

Introduction

Synovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicans-induced septic arthritis is largely unknown.

Methods

Primary cultures of rat synovial fibroblasts were infected with C. albicans (ATCC90028). Immunocytochemistry, western blotting, and RT-PCR were performed to assess cyclo-oxygenase 2 induction. Phosphorylation of extracellular-regulated kinase (ERK1/2) following infection in the absence or presence of U0126 was assessed by western blotting whilst prostaglandin E2 production was measured by ELISA. Nuclear factor κB (NFκB) translocation was evaluated by an electrophoretic mobility shift assay.

Results

Infection of synovial fibroblasts with C. albicans resulted in cyclo-oxygenase 2 expression and prostaglandin E2 production. Cyclo-oxygenase 2 expression and prostaglandin E2 production was dependent upon extracellular-regulated kinase 1/2 phosphorylation, associated with activation of NFκB and significantly elevated in the presence of laminarin, an inhibitor of dectin-1 activity. Synovial fibroblasts adjacent to C. albicans hyphae aggregates appeared to be the major contributors to the increased levels of cyclo-oxygenase 2 and phosphorylated extracellular-regulated kinase 1/2.

Conclusions

C. albicans infection of synovial fibroblasts in vitro results in upregulation of cyclo-oxygenase 2 and prostaglandin E2 by mechanisms that may involve activation of extracellular-regulated kinase 1/2 and are associated with NFκB activation.     

Anti-inflammatory effects of spermidine in lipopolysaccharide-stimulated BV2 microglial cells

Background

Spermidine, a naturally occurring polyamine, displays a wide variety of internal biological activities including cell growth and proliferation. However, the molecular mechanisms responsible for its anti-inflammatory activity have not yet been elucidated.

Methods

The anti-inflammatory properties of spermidine were studied using lipopolysaccharide (LPS)-stimulated murine BV2 microglia model. As inflammatory parameters, the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were evaluated. We also examined the spermidine''s effect on the activity of nuclear factor-kappaB (NF-κB), and the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways.

Results

Pretreatment with spermidine prior to LPS treatment significantly inhibited excessive production of NO and PGE₂ in a dose-dependent manner, and was associated with down-regulation of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Spermidine treatment also attenuated the production of pro-inflammatory cytokines, including IL-6 and TNF-α, by suppressing their mRNA expressions. The mechanism underlying spermidine-mediated attenuation of inflammation in BV2 cells appeared to involve the suppression of translocation of NF-κB p65 subunit into the nucleus, and the phosphorylation of Akt and MAPKs.

Conclusions

The results indicate that spermidine appears to inhibit inflammation stimulated by LPS by blocking the NF-κB, PI3K/Akt and MAPKs signaling pathways in microglia.     

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Macrophage Colonization by Salmonella enterica Serovar Typhimurium

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is an anti-inflammatory downstream product of the cyclooxygenase enzymes. It has been implicated to play a protective role in a variety of inflammatory mediated diseases, including rheumatoid arthritis, neural damage, and myocardial infarctions. Here we show that 15d-PGJ₂ also plays a role in Salmonella infection. Salmonella enterica Typhimurium is a Gram-negative facultative intracellular pathogen that is able to survive and replicate inside phagocytic immune cells, allowing for bacterial dissemination to systemic sites. Salmonella species cause a wide range of morbidity and mortality due to gastroenteritis and typhoid fever. Previously we have shown that in mouse models of typhoid fever, Salmonella infection causes a major perturbation in the prostaglandin pathway. Specifically, we saw that 15d-PGJ₂ production was significantly increased in both liver and feces. In this work we show that 15d-PGJ₂ production is also significantly increased in macrophages infected with Salmonella. Furthermore, we show that the addition of 15d-PGJ₂ to Salmonella infected RAW264.7, J774, and bone marrow derived macrophages is sufficient to significantly reduce bacterial colonization. We also show evidence that 15d-PGJ₂ is reducing bacterial uptake by macrophages. 15d-PGJ₂ reduces the inflammatory response of these infected macrophages, as evidenced by a reduction in the production of cytokines and reactive nitrogen species. The inflammatory response of the macrophage is important for full Salmonella virulence, as it can give the bacteria cues for virulence. The reduction in bacterial colonization is independent of the expression of Salmonella virulence genes SPI1 and SPI2, and is independent of the 15d-PGJ₂ ligand PPAR-γ. 15d-PGJ₂ also causes an increase in ERK1/2 phosphorylation in infected macrophages. In conclusion, we show here that 15d-PGJ₂ mediates the outcome of bacterial infection, a previously unidentified role for this prostaglandin.     

Effects of 15-deoxy-∆<Superscript>12,14</Superscript>-prostaglandin J<Subscript>2</Subscript> on the production of IL-8 and the expression of Toll-like receptor 2 in human primary keratinocytes stimulated with lipopolysaccharide

15-deoxy-∆^12,14-prostaglandin J₂ (15d-PGJ₂) is an anti-inflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We investigated the effects of 15d-PGJ₂ on the production of IL-8 and on the expression of Toll-like receptors (TLRs) 2 in human primary keratinocytes stimulated with lipopolysaccharide (LPS). Cell proliferation was analyzed using the MTT assay, TLR2 and -4 mRNA expression was detected by RT–PCR, and IL-8 production and NF-κB p65 activities were determined by ELISA. LPS and 15d-PGJ₂ did not influence the proliferation rate at low concentrations (0.5 and 2.0 μM) in keratinocytes, and showed toxicity at high concentrations (5.0 μM). LPS, compared with control, induced the expression of TLR2 mRNA, increased IL-8 production, and enhanced NF-κB activity. 15d-PGJ₂ decreased TLR2 mRNA, increased IL-8 production, and suppressed NF-κB activity. Costimulation with LPS and 15d-PGJ₂, compared with LPS stimulation alone, decreased TLR2 mRNA (1.8-fold), increased IL-8 production (1.8-fold at 0.5 μM and 3.7-fold at 2.0 μM), and inhibited NF-κB activity (3.3-fold at 0.5 μM and 5.1-fold at 2.0 μM). TLR4 mRNA was not expressed in primary keratinocytes. These results suggest that 15d-PGJ₂ suppresses TLR2 expression and that it up-regulates the production of IL-8 by inhibiting the NF-κB signaling pathway in primary keratinocytes. Thus, 15d-PGJ₂ can have both anti- and pro-inflammatory effects, and 15d-PGJ₂-mediated IL-8 up-regulation is related to the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E₂ synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF_1α and PGE₂ in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF_1α and PGE₂ peaked 24 hours after stimulation with IL-1β; the induction of PGE₂ was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE₂ level than on 6-keto-PGF_1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ₂ partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ₂ were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM^? analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE₂ production, whereas 15d-PGJ₂ inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE₂ synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ₂ strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ₂ occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ₂.     

Myocardial Creatine Levels Do Not Influence Response to Acute Oxidative Stress in Isolated Perfused Heart

Background

Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury.

Objectives

To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse.

Methods and Results

CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo ¹H^–MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H₂O₂ 0.5 µM (30 min), or the anti-neoplastic drug doxorubicin 15 µM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCδ expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wild-type controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction.

Conclusions

Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity.